The presence of insulin resistance in normoglycemic subjects with a strong family history of type 2 diabetes attests to its hereditary nature. The insulin receptor signaling system is a likely focus of insulin resistance. In type 2 diabetes, there are defects in insulin receptor signaling that include decreased insulin stimulation of insulin receptor and IRS-1 tyrosine phosphorylation and the association of PI 3-kinase with IRS-1. In diabetics one bout of exercise improves insulin receptor tyrosine phosphorylation but does not increase the association of IRS-1 with PI 3-kinase, suggesting that the latter abnormality may be intrinsic or hereditary. In normal glucose tolerant subjects with a family history of type 2 diabetes, insulin resistance is associated with a defect in insulin stimulated IRS-1 tyrosine phosphorylation and association of PI 3-kinase activity with IRS-1. In contrast, insulin-stimulated tyrosine phosphorylation of the insulin receptor is normal. Taking these findings together, we hypothesize that a defect in IRS-1 function in skeletal muscle plays a key role in insulin resistance and type 2 diabetes. Specifically, we propose: 1) To identify sites of serine/threonine phosphorylation of endogenous IRS-1 in human skeletal muscle in vivo. We will use recombinant IRS-1 proteins and synthetic peptides to identify candidate serine/threonine phosphorylation sites phosphorylated by known kinases or human skeletal muscle lysates and subcellular fractions. Sites will be identified by consensus sequence analysis, two-dimensional phosphopeptide mapping, MALDI-TOF and HPLC-ESI/MS/MS analysis. Based on these results, we will target the serine/threonine phosphorylation sites that are held in common by recombinant known kinases and human muscle lysates and fractions. Then we will identify endogenous IRS-1 phosphorylation sites by HPLC-ESI/MS/MS and MALDI-TOF analysis of immunoprecipitated IRS-1 from muscle biopsies taken from subjects basally and during insulin infusion (euglycemic hyperinsulinemic clamp). 2) To determine the role of serine/threonine phosphorylation of IRS-1 in insulin resistance. 3) To determine if common mutations in IRS-1 are associated with defects in IRS-1 function in nondiabetic Mexican American individuals who have a strong family history of type 2 diabetes and decreased insulin-stimulated association of IRS-1 with PI 3-kinase. 4) To use DNA microarray technology to search for genes that are abnormally expressed in normal glucose-tolerant subjects with a strong family history of type 2 diabetes. We will test the hypothesis that these subjects respond differently to experimental conditions that provoke insulin resistance.